Cytokeratins (hereinafter, referred to as “CK”) are proteins forming fibrous backbone of a cell and form a family of at least 20 genes. As such cytokeratins, human CK18, human CK19, human CK20 and the like are known. CKs are expressed in epithelial cells.
For example, previous reports described that human CK18 is expressed not only in normal tissues such as mammary gland, lung, large intestine, stomach but also in cancerous tissues, while human CK18 is not expressed in lymph nodes which are not epithelial tissues. As to human CK19, previous reports described that it is expressed in lung cancer, stomach cancer, breast cancer, pancreatic cancer, prostate cancer and the like, and difference in expression level of human CK19 is observed between normal tissues and cancerous tissues. As to human CK20, previous reports described that it is expressed in colon cancer, stomach cancer, Merkel cell cancer, gynaecologic mucus cancer, transitional cell cancer, pancreatic cancer and bile duct cancer, and also in this case, difference in expression level of human CK20 is observed between normal tissues and cancerous tissues.
In consideration of the above facts, it is possible to know presence/absence of metastasis of cancer by examination of presence/absence of expression of human CK18, human CK19 and/or human CK20 in tissues such as lymph nodes. Also it is possible to know presence/absence of metastasis of cancer by searching for those exhibit difference in expression level between normal tissues and cancerous tissues.
A cancer cell leaving the primary lesion site will metastasize around the body through the blood stream and lymphatic system. In a surgery of cancer, accurate detection of metastasis and proper treatment based on the extent of metastasis are required so as to remove lesions as reliable as possible. For this reason, intraoperative diagnosis of cancer metastasis to lymph nodes has great significance. For example, in the case of breast cancer, the range to be removed is getting smaller for improving the QOL, and diagnosis for lymph node metastasis during the surgery can be significant guidelines for determining the minimum of lymph node dissection. In the case of esophageal cancer, detection of a site where lymph node metastasis occurs may give guidelines for selection and determination of operative procedures including ventrotomy, thoracotomy and collar incision. In the case of prostate cancer, presence of lymph node metastasis will give guidelines for decision to conduct hormonal therapy while stopping the prostatectomy. Also in the case of stomach cancer, it will give guidelines for selecting an operative procedure and therapeutic strategy after the surgery. Considering the burden on a patient, intraoperative diagnosis for cancer metastasis should be conducted rapidly.
One procedure for diagnosing cancer metastasis to lymph nodes is detecting CK proteins which are tumor markers. For example, this is achieved by freezing a resected lymph node and staining a section of the frozen tissue. However, this procedure is accompanied with a risk that micrometastasis is overlooked because only the information about the section is relied on.
Recent development in gene analysis technique has enabled effective cancer diagnoses through detection of expression of tumor marker genes. For example, PCR technique enables a target DNA fragment to be amplified to several hundreds times by repetition of dissociation of DNA strands to a single-stranded DNA, binding of primers sandwiching a specific region in the DNA strand, and synthesis of DNA by DNA polymerase (see JP-A 61-274697), and can be used as a high-sensitive analyzing technique for nucleic acids in various types of sample. For example, since the PCR technique can analyze nucleic acids in samples obtained from animal body fluids or tissues, it is useful for diagnosis of infection diseases, genetic diseases, cancers and the like.
For detection of RNA, RT-PCR technique can be used. the RT-PCR technique involves extracting RNA, for example, from tumor tissues; synthesizing cDNA by the help of reverse transcriptase (RT) using oligo(dT) or random hexamer as a primer; amplifying the cDNA using PCR technique for detection. The exemplary case of diagnosis of fibroblast tumor using the RT-PCR technique has been reported (Hokkaido Igaku Zasshi p. 135-141, Vol. 66(2), (1991)). The RT-PCR makes it possible to detect expression of mRNA of CK from ablated tissues, so that the problem that cancer metastasis is overlooked can be avoided to some extent. In the field of diagnosis of tumor or cancer, such nucleic acid amplifying methods have been put into practical use (“Kanai's manual of clinical laboratory medicine” the 31st ed., pp. 1314, KANEHARA & Co., LTD., published on Sep. 20, 1998).
However, the PCR technique necessitates an operation for denaturing a template DNA from double-strand DNA to single-strand, as well as requires repetitive amplifying reactions under plural temperature conditions. Furthermore, in general, it takes about two hours to obtain a detectable amplified product, so that it was not desirable as an intraoperative test that requires rapidity.
As a DNA amplifying method other than the PCR technique, LAMP method has been reported (refer to International Publication WO00/28082). The LAMP method is a gene amplifying method using a plurality of primers including a primer that forms a hairpin structure at a terminal of the amplified product as the strand displacement reaction proceeds. In an initial reaction, using two kinds of inner primers (FIP, RIP) and two kinds of outer primers (F3 primer, R3 primer) and a strand displacing DNA polymerase, a dumbbell-like structure having a single-strand loop at each end is synthesized from a template DNA. Starting from this structure, the amplification cycle carried out so that extension and synthesis of DNA proceed on the DNA itself as a temperate, from the 3′ end of this structure. The amplified product comprises a repeated structure of plural units, and each unit comprises a set of complementary regions in the same strand that forms a region to be amplified sandwiched between the primers wherein two nucleic acids have inverted base sequences. The LAMP method does not require the operation for denaturing a template DNA from double strand to single strand by heating, and is characterized by continuous process of amplification under a constant temperature (refer to Bio Venture, Vol. 1, p. 109-115 (2001) and BIO INDUSTRY, Vol. 18, No. 2, p. 15-29 (2001)). When the template is RNA, it is possible to synthesize the starting structure in a similar manner by adding a reverse transcriptase to the composition of the reaction mixture, whereby amplification can be proceeded (RT-LAMP method). According to the LAMP method, a sufficient amount of amplified product for detection can be obtained in about 30 minutes. Therefore, the time required for detection is reduced, so that it can be applied for diagnosing cancer metastasis to lymph nodes for the purpose of rapid determination of the therapeutic strategy, for example. Furthermore, since a result can be acquired rapidly, application to the intraoperative diagnosis is also expected.
The basic concept of primers applied to the LAMP method can be found in International Publication No. WO00/28082 and International Publication NO. WO02/24902.
Primers and probes to be used in PCR for detection of human CK18 have been already reported (refer to Gene, 159(1), p. 43-47(1995)). Likewise, primers or probes to be used in PCR for detection of human CK19 have also been reported (refer to U.S. Pat. No. 6,203,992 and Breast Cancer Research and Treatment 60, p. 143-151(2000)) Also primers or probes to be used in PCR for detecting human CK20 have been reported (refer to British J. of Cancer, 77(8), p. 1327-1332(1998) and British J. of Cancer, 82(1), p157-160 (2000)).
However, nobody have reported primers to be applied to LAMP method intended for detection of human CKs, and there is a need to develop such primers. In addition, as for the primers to be applied to other nucleic acid amplifying means, there is a need to construct a new primer or a primer set that is useful for detection in addition to known primers.
(Document Of Conventional Art)
Patent document 1: International Publication No. WO00/28082
Patent document 2: International Publication No. WO02/24902
Patent document 3: U.S. Pat. No. 6,203,992
Non patent document 1: Bio Venture, Vol. 1, p. 109-115 (2001)
Non patent document 2: BIO INDUSTRY, Vol. 18, No. 2, p. 15-29 (2001)
Non patent document 3: Gene, 159(1), p. 43-47(1995)
Non patent document 4: Breast Cancer Research and Treatment 60, p. 143-151(2000)
Non patent document 5: British J. of Cancer, 77(8), p. 1327-1332(1998)
Non patent document 6: British J. of Cancer, 82(1), p157-160 (2000)